Bioremediation As a Tool for Resiliency in Oil Spills

Bioremediation as a Tool for Resiliency in Oil Spills

By: Caitlin G. Bergman, Elena R. Eberhart, Mackenzie A. Garner, Atticus H. Hamilton, and Claire M. Bredar

Coach: Chris Backstrum

South Anchorage High School 13400 Elmore Road Anchorage, Alaska 99504 Contact person: Caitlin Bergman at [email protected]

“This paper was written as part of the Alaska Ocean Sciences Bowl high school competition. The conclusions in this report are solely those of the student authors.”

Bioremediation as a Tool for Resiliency in Oil Spills

Abstract

With an increase in boating, offshore drilling, and transportation of oil, coastal communities are in need of a plan to clean up waterways in the event of an offshore oil spill. Manual removal of oil is a critical first step and when paired with bioremediation as a secondary method the highest possible success of oil breakdown at a spill site can be achieved. Unlike dispersants, bioremediation is a clean way to speed up the degeneration of oil in waterways. According to research done by Gordon in 1994, given that proper nutrients are present, an oil spill that was estimated to be cleaned by natural conditions in

5-10 years could be cleaned in 2-5 years with the use of bioremediation. Alcanivorax borkumensis, an oil-degrading microbe, was added to water with heavy fuel oil. After two months the water was clean enough to return back into the seas (Golyshin, n.d.). This bacteria is found in marine ecosystems and can absorb and digest linear and branched alkanes that are found in crude oil and its products (Rojo, 2009). Community resilience to oil spills would involve establishing a preparedness plan with a system in place to quickly implement bioremediation methods. Establishing oil spill preparedness practices in coastal communities will be critical to reduce impact from oil. This system would incorporate culturing tanks, quantities of hydrocarbonoclastic bacteria in pellet form, and the means to transport and apply in the field.

Introduction

When oil spills occur, they can cause major damage to the environment. There have been many oil spills in Alaska (Table 1), with devastating environmental effects.

Many marine mammals have been killed, fisheries have been closed, and native populations have lost their food source as a result of oil spills (Amadeo, 2015). It is very important for communities to have a plan for what to do in the event of an oil spill.

Bioremediation uses oil-degrading bacteria to clean up the spill. Bioremediation has already been used in many oil spills, including the spill in Prince William Sound. Adding bacteria and nutrients to oil spills will significantly increase the degradation of the oil, with less environmental impacts than other cleanup methods. This will help communities to be more resilient, and recover more quickly after the spill.

Bioremediation

Bioremediation is the use of naturally occurring or introduced microorganisms or other forms of life to consume or break down environmental pollution in order to clean up the polluted area (Collins English Dictionary, 2015). Bioremediation is an alternative cleanup action that is safer for the environment than other chemical or physical solutions.

The microorganisms involved with bioremediation are either already living in the affected environment or they are brought into the environment (Cornell, 2009). If the chosen bioremediation process involves the naturally occurring microorganisms in the ocean environment, then the group organizing the bioremediation cleanup would add

1 nutrients to the water to help boost population growth of the native species of bacteria.

This was used in Alaska, during the Exxon Valdez oil spill in Prince William Sound.

Fertilizer was added to beaches in order to supply nutrients to naturally occurring microbes in the water. The oil in fertilized areas biodegraded about two times faster than untreated controls (Pritchard et al., 1992). The other main method of bioremediation is to take cultured microorganisms and introduce them into an oil spill and then continue to feed them, making the population grow.

Composition of Crude Oil

Crude oil is made up of four main elements. It usually contains 84% to 87% carbon, 11% to 14% hydrogen, 0.1% to 8% sulfur, 0.1% to 1.8% nitrogen, and 1% to

1.5% oxygen (Lyons, 2005). Although there are sodium, nitrogen, and oxygen compounds in oil, the most common molecules are hydrocarbons. There are three main groups of hydrocarbon molecules in crude oil; aromatics, naphthenes, and alkanes

(Australian Institute of Petroleum, 2013).

The most difficult hydrocarbons for bacteria to biodegrade are aromatic compounds. Aromatic compounds are double-bonded carbon rings. Some of them also have attached chains of hydrocarbons. Very small (one or two ring) aromatics evaporate off of a spill or can be biodegraded. Larger aromatics, however, resist biodegradation, and can persist in the area of a spill for a long time (American Academy of Microbiology,

2011). The only way that they can be broken down is by photo oxidation, or degradation by U.V. light. Aromatics are also the most toxic compounds in crude oil (NOAA, 2015).

2 Naphthenes are single-bonded, saturated hydrocarbon rings. Naphthenes can be biodegraded more easily than aromatics, but not as quickly as alkanes because they contain more bonds (El-Nemr, 2006).

Alkanes are straight or branched saturated hydrocarbons. They only contain single bonds, which is ideal for microbial degradation because it does not take much energy to break apart the molecules, compared to double-bonded molecules. They can be solids, liquids, or gases, depending on the number of carbon atoms they contain. Alkanes with one to four carbon atoms are gases, also called volatile compounds. During an oil spill, these compounds evaporate off of the slick and into the air. Alkanes with five to sixteen carbon atoms are liquids. These form most of the oil slick. They can be degraded relatively quickly by bacteria. The smaller the chain, the easier it is for bacteria to break it down. Alkanes with more that sixteen carbon atoms are solids, and are difficult for bacteria to break down.

The characteristics of crude oil differ from place to place. Alaskan North Slope crude oil has a relatively high viscosity, and forms an emulsion with water very quickly

(NOAA, 2015). This makes it extremely important to respond quickly to spills made up of this type of oil.

With the exception of aromatic compounds, all hydrocarbons in crude oil can be degraded by bacteria. This makes bioremediation a very important method for cleaning up oil spills. In time, naturally occurring bacteria will completely break down a spill.

However, they cannot do this quickly enough to prevent damage to the ecosystem, unless bioremediation methods are used to speed up their growth.

3 Alcanivorax borkumensis

Alcanivorax borkumensis is a marine bacteria that can absorb and digest linear and branched alkanes that are found in crude oil and its products (Rojo, 2009). A. borkumensis is a gram-negative bacteria, meaning that the bacteria has an outer membrane of lipopolysaccharides, unlike gram positive bacteria who do not possess this layer. It is also a rod-shaped bacterium that is aerobic (oxygen reliant) (Biello, 2006). A. borkumensis is included in the genus Bacillus, which is a genus for rod-shaped bacterium and is in the class Gammaproteobacteria, meaning that it is a scientifically important bacteria (Kostka et al., 2011). Since A. borkumensis occurs naturally in unpolluted waters all over the world (including freshwater), it has to have a source of energy. A particular study has found that strains of two of the most abundant cyanobacteria in the ocean

(Prochlorococcus and Synechococcus) produce and accumulate hydrocarbons, particularly alkanes C15 and C17 (PNAS, n.d.). These alkanes are the energy source for

A. borkumensis in unpolluted water. A. borkumensis naturally flourishes after an oil spill because there is a more abundant source of energy that can sustain a larger population

(Kimes, 2014). A. borkumensis also participates in wastewater treatment by being foamed by Nocardia spp. (Shamoon, n.d). A borkumensis breaks apart the bonds in hydrocarbons in oil that have been exposed to the sea, using enzymes and oxygen found in the seawater

( Biello, 2010).

A. borkumensis creates enzymes AlkB1 and AlkB2 (Beilen, 2004). AlkB1 is involved with the direct reversal of alkylation damage, specifically in single-stranded

DNA ( Dinglay, 2000). AlkB1 hydroxylases alkanes with 5 to 12 carbons, and AlkB2 hydroxylases alkanes with 8 to 16 carbons (Rojo, 2009). The chain lengths with the most

4 A. borkumensis growth are 14 to 19 carbon chains (Naether et al., 2013). A. borkumensis is able to outcompete other hydrocarbonoclastic species of bacteria because it can break down such a wide range of alkane chains (Hara, Akihiro, Kazuaki Syutsubo, & Shigeaki

Harayama, 2003). A. borkumensis cannot consume sugars or amino acids as a source of energy, unlike most other bacteria (Yakimov, Michail M., et al, 1998).

A. borkumensis Genetics

The Alcanivorax Borkumensis bacterium contains a single ringed chromosome free floating in the cytoplasm. This single stranded circular chromosome contains islands.

It is these islands that code for the bacteria’s oil degrading enzymes. The islands are made up of genes that code for different enzymes. These genes are mobile, meaning that they can change their position in the chromosome, also allowing the islands to transfer to other bacteria of different species through horizontal gene transfer. In this way, non-oil degrading microbes can develop the enzyme creation by horizontal gene transfer with A. borkumensis. Then, they are able to degrade oil. Alcanivorax borkumensis has in its SK2 genome a number of regions, and one such island contains a cluster of 40 genes which code for cell surface biosynthesis. The second island holds a complete cluster of genes for alkane degradation. This cluster is called alkSB1GJH. The enzyme produced, ALKB

1, oxidises medium length alkanes, with 5 to 12 carbons.

Culturing A. borkumensis

When culturing A. borkumensis it is imperative that a scientist uses a medium that supplies nitrogen, phosphorus, oxygen, and sulfur or sulfate (Schneiker, 2006). During the degradation of hydrocarbons by A. borkumensis these minerals are used to boost the growth rate of the microbe. The more of these nutrients that are present in an

5 environment, the larger the bloom of A. borkumensis becomes. This therefore requires a supplement for oil nutrients when culturing the A. borkumensis bacterium.

Along with sodium pyruvate, this mineral supplementation is achievable by the use of sodium chloride, sodium sulfate, sodium bromide, sodium hydrocarbonate, hydrogen boron oxide, hydroxypropanesulfonic acid (Naether et al., 2015). This form of medium can be turned into a pellet containing the bacterium. The pellet can be formed by culturing the bacterium in a water shake culture (which is inoculating a tube with the warm liquid culture medium and shaking up the tube so even distribution of the contents is achieved.) (Naether et al., 2015) (The Free Dictionary). Once the liquid has been adequately shaken and mixed, the resulting fluid is deposited into into a centrifuge at four degrees celsius for fifteen minutes (Sabirova, Ferrer, Regenhardt, Timmis & Golyshin,

2006). Finally the resulting pellet is freeze dried and can be stored at -20 to -70 degrees

Celsius, or -4 to -94 degrees Fahrenheit (Sabirova et al., 2006). To rehydrate, take the pellet and hydrate it with nutrient-rich seawater or another recommended medium. Then, transfer the rehydrated pellet into a tube with the rehydration medium and mix until clumps are absent. Finally the medium can either be transferred to an agar slant (The

Essentials of Life Science Research, 2014) or incubated in solution at 30 degrees celsius

(Naether et al., 2015).

Other Hydrocarbonoclastic Bacteria

Although A. borkumensis is the most efficient oil-degrading bacteria, there are many different species that work on different parts of the alkane chain length. A few main species associated with the degradation of oil would be Marinobacter,

Pseudomonas, and Acinetobacter (Kostka et al., 2011). A sample from the Deepwater

6 Horizon spill in the Gulf of Mexico showed a wide variety of different kinds of hydrocarbonoclastic bacteria, but A. borkumensis was most abundant (Kostka et al.,

2011).

Another natural action is horizontal gene transfer, the transfer of genes without asexual or sexual reproduction (Gyles & Boerlin, 2014) (Figure 1). In a study done on

Pseudomonas, the dominant polycyclic aromatic hydrocarbon degrading bacteria, the bacteria used horizontal gene transfer to switch its genes on transmissible plasmids which in turn spread the genes throughout the indigenous species of bacteria (Ma, Wang &

Shao, 2005).

Figure 1 An example of horizontal gene transfer in bacteria, demonstrating the transmissible plasmid and how it moves between bacteria (Berkeley, n.d.)

Effects of Bioremediation

Bioremediation is a fairly new endeavor, with little known about the possible side effects. So far it is understood that by using bioremediation techniques, oil spills can be cleaned up more cheaply, safely, and more efficiently than oil spills cleaned solely with manual mechanisms and dispersants. However, there are environmental impacts to be

7 taken into consideration, like the depletion of oxygen in the areas where bioremediation is taking place.

There are high amounts of oil being deposited into the oceans every year (Figure

3). As with many oil spills, the Exxon Valdez oil spill was difficult to clean up. After a period of time had passed, bioremediation was used. Before bioremediation was implicated in 1991, there were still vast amount of oil present. Based on a study done by the National Oceanic and Atmospheric Administration (NOAA) in 2001 and 2003, 97.8% of the seventeen sites tested, all of which were moderately or heavily oiled in 1989, did not contain or contained very little oil after the use of bioremediation techniques. Based on those results, there was an estimated 22% decline of oil in the area from 1991-2001

(Short et al., 2004). After 2001, the degradation of oil dropped to about 4% per year due to the oil being weathered and more sequestered (Short et al., 2007).

Oxygen depletion

Figure 3 Average annual contribution to oil in the ocean occurs in areas where (1990-1999) from major sources of petroleum in kilotonnes (Ocean Studies Board, 2013) bioremediation is used.

The amount of oxygen that would be depleted depends on the amount of oil in the water and how many hydrocarbon-degrading microbes are present. Due to high spill rates and marine traffic in the Gulf of Mexico, large populations of oil degrading microbes are

8 present. There was a 30% depletion of oxygen in the water near the oil in this region

(Atlas & Hazen, 2011). With proper nutrients, oil spills can be cleaned at a rate of 1.2% each day by using bioremediation techniques (Atlas & Hazen, 2011). Hypoxia was not detected in these areas. This means that while there was a depletion of 30% of the oxygen in the water, after the population of hydrocarbon degenerating microbes died, photosynthetic organisms were able to produce more oxygen and return the levels back to normal after a series of years had past.

Dispersants and Biosurfactants

Dispersants are chemicals that are used to break up oil spills. They reduce interfacial tension between oil and water, and break up the oil slick into small droplets.

Each molecule of dispersant has a polar end and a nonpolar end (Hervé, 2010). This allows it to interact with both water and oil. Molecules of surfactant surround droplets of oil, which disperse into the water. Dispersants remove the oil slick from the surface, but also spread oil throughout the water column, which allows the spill to affect more organisms. (Laleian & Azwell, 2011). A. borkumensis is most active in surface waters, although it can be found at most depths. If dispersants are added to the oil spill, it will slow down bioremediation.

More dispersants were used during cleanup of the spill in the Gulf of Mexico than for any other oil spill in the United States. It was also the first place that dispersants were released underwater, directly into the flow of oil (Kujawinski et al., 2011). Now, as a result of both dispersants and oil, the Gulf of Mexico is experiencing serious environmental impacts, such as diminishing sea turtle and shellfish populations

(Cameron, 2015).

9 Biosurfactants are molecules that are produced by bacteria, and have the same properties as commercially produced dispersants. Many oil-degrading bacteria, including

A. borkumensis, produce biosurfactants to facilitate the diffusion of hydrocarbons into the cell, and also to break up the oil slick so that it has more surface area for them to feed on

(Karanth, Deo & Veenanadig, 1999). Biosurfactants are being produced naturally as oil is broken down by bacteria, during bioremediation of the oil spills.

The biosurfactants that are produced naturally during bioremediation can replace commercially produced dispersants in oil spill remediation. They have the same properties, but are organic and non-toxic. Our research shows that dispersants should not be added to oil spills, except as a last resort.

Bioremediation as a Tool for Resiliency in Oil Spills

A deepwater oil spill is undoubtedly a catastrophic event.

Almost immediately, sensitive areas can be threatened, and it is ultimately up to humans to remedy this situation. There are various mechanical methods that can be taken to protect the environment, Figure 2 Oil skimmer in the Gulf of Mexico including containment, skimming, and in removing surface oil, in April 2010. situ burning. The main goal of taking these mechanical measures is to prevent oil slicks from threatening sensitive regions and to prevent oil from reaching shorelines, where it inevitably becomes more difficult to clean.

10 In the event of a large scale oil spill in Alaskan waters, reminiscent of the Exxon

Valdez spill, prompt action must be taken. If oil reaches shorelines, there are a few options to attempt to clean beaches. First, trained clean up crews employ shoreline flushing, which involves spraying water, at a specific temperature and pressure, onto oil contaminated surfaces in order to refloat or remove the slick. Next, industrial-sized vacuums and specialized sorbents can be used to remove oil. Most often in remote locations, like Alaskan beaches, an oil slick coming ashore means manual recovery teams must be deployed to clean shorelines with rakes, buckets, and other hand tools. (National

Oceanic and Atmospheric Administration, 2015). Manual recovery and clean up is extremely laborious and inefficient work. In addition, once oil reaches the shoreline, it exposes a whole new ecosystem to potentially lethal contamination.

In order to avoid these severe consequences, mechanical measures must be taken while the oil is still afloat. Immediately after an oil spill has occurred, booms should be positioned surrounding the slick, containing it and massing it together. Booms are physical barriers, often made of plastic or metal, that float on the water (NOAA, 2015).

Sorbent boom and hard boom are the two most useful categories. Hard boom is made up of a floating plastic top and a weighted “skirt” that rests below the water, allowing it to contain oil especially effectively and with more longevity than other booms (NOAA,

2015). Sorbent boom does not contain the “skirt” that hard boom does, but instead is equipped with material that absorbs oil, creating a safe barrier between the slick and clean water. (NOAA, 2015). Additionally, boat systems called skimmers (Figure 2) work by brushing oil off the surface of the water and collecting it. They should be used to aid

11 in the mechanical removal of the oil in the early stages of a spill response, when the sheer mass of an oil slick is threatening to communities and ecosystems.

Our research suggests a relatively cheap, cost-effective method of bioremediation should be a feasible next step. Coastal communities that face the threat of a large scale oil spill would already have in place the ability to cultivate large quantities of A. borkumensis with a lag time of approximately seventy-two hours. A system should be established in these communities, made up of a 5,000 gallon tank, 2,300 pounds of A. borkumensis in pellet form, and water heaters. In order for incubation of A. borkumensis to occur in a timely manner, the frozen pellets must be melted and dispersed in 30 degree

Celsius seawater. The pellets should be placed in the 5,000 gallon tank along with approximately 132 gallons of sodium pyruvate nutrient (Hiral, 2015). From there, the tank should be filled with seawater in order to create a medium for the bacteria to grow.

Industrial sized water heaters should be placed in the tank until the water reaches the incubation temperature of 30 degrees Celsius.

From the tank, the bacteria should be introduced into the spill. At the same time nutrients, especially nitrogen and phosphorous, should be added to the spill (Pritchard et al., 1992). The addition of nutrients will mean that there will be no limiting factors for the bacteria, and they will consume the oil faster. Once the oil is gone, they will have a much smaller energy source and will begin to die off.

At the same time of physical removal, oil spill responders should start the process of culturing the pellets of A. borkumensis in a tank of water mixed with sodium pyruvate to supplement the bacterias carbon source. Importance of early action cannot be

12 emphasized enough, as the more time that passes, the more the oil will disperse, making it harder for clean up crews to collect oil (International Tanker Owners Pollution

Federation, 2012). Although oil may not be visible if allowed to disperse, it is in no way absent.

Conclusion

It is extremely important that coastal communities have a plan in place for when an oil spill occurs. Having a response plan that can be implemented immediately after the spill, and will not cause much damage to the environment, will allow the community to recover as quickly as possible. Bioremediation satisfies these requirements. It does not have many negative effects on the environment. A. borkumensis lives naturally in all oceans, so adding more does not affect the ecosystem as much as introducing a foreign species. Bioremediation is much less toxic than dispersants, and does not destroy the landscape, like manual recovery. With the right preparation, it can be started within 72 hours of the spill. Nutrient addition has been proven to be effective in Alaskan waters, such as the Exxon Valdez oil spill, and adding microbes along with nutrients would speed up the process further. Our research shows that bioremediation, specifically the addition of microbes into an oil spill, is an effective and environmentally friendly method to clean up oil spills. A bioremediation system would help communities, both human and animal, to be resilient after an oil spill.

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